PROJECT SUMMARY/ABSTRACT Toxoplasma gondii with the ability to use foodborne, zoonotic, and congenital routes of transmission is an apicomplexan parasite that can cause severe infectious disease in the immunocompromised human population. A few antibiotics are commercially available to treat Toxoplasma infections. However, their strong side effects and teratogenicity limit their use in certain human populations. Inhibition of fundamental nutrient metabolism specific to this parasite will define new drug targets and assist the development of novel drugs to manage T. gondii infection. Our previous studies revealed that Toxoplasma encodes an ortholog of Plasmodium chloroquine resistance transporter (TgCRT), and localized it in the digestive vacuole, termed the Vacuolar Compartment/Plant-Like Vacuole (VAC/PLV, VAC hereafter). Our preliminary data found that the TgCRT-deficient parasites swelled their VACs ~15-fold. These results led to our central hypotheses: (1) the TgCRT serves as a polyspecific transporter to regulate the VAC physiology and function in Toxoplasma, and (2) the TgCRT cooperates with a multidrug resistance transporter-like protein in the VAC to mediate nutrient export, thereby adjusting the microenvironment within the VAC. Towards these hypotheses, we have revealed that the swollen VAC disrupts the parasite?s endolysosomal system and decreases transcript and protein abundances of VAC-associated proteases. We discovered that inhibition of proteolysis within the VAC shrinks its swollen phenotype in TgCRT-null mutant. We have also identified that a Toxoplasma ortholog of Plasmodium multidrug resistance transporter (TgMDR) is localized in the VAC and significantly increased at the level of transcription in the parasites when TgCRT is absent. Last, we determined that TgCRT transports chloroquine by heterologous expression of TgCRT in yeast, suggesting that TgCRT is indeed a functional transporter and providing an amenable system to understand the native functions of TgCRT and other VAC-localizing transporters. Guided by our compelling preliminary studies, we propose three specific aims to characterize the native functions of TgCRT and TgMDR, and how they functionally interact together to regulate VAC physiology and function by serving as nutrient transporters: (1) Quantify the physiological environment within the VAC; (2) Measure the transport of small nutrient solutes by CRT; and (3) Determine the functional relationship between TgCRT and TgMDR in the regulation of the VAC morphology and physiology. Our proposed research will broadly impact the field by characterizing the molecular mechanisms by which Toxoplasma parasites regulate the physiology and function of their digestive vacuoles. Our studies will also help comprehend the native functions of TgCRT and TgMDR, and such knowledge can be generalized to expand understanding of their orthologs in other apicomplexan parasites and organisms.